GSM8047383: ILCs - No Abx; Mus musculus; RNA-Seq
The whole colon from mice was dissected and opened longitudinally, followed by washing with PBS. Tissues were then left in buffer 1 (HBSS with 25 mM HEPES and 5% FBS) until harvesting all the mice. Colon tissues were incubated at 37°C temperature with gentle shaking for 40 min to remove the epithelial layer into 40 ml Buffer 2 (HBSS with 15 mM HEPES, 5 mM EDTA, 10% FBS, and 1 mM DTT). Tissue sections were cut into small pieces with a fine scissor and incubated into 5 ml digestion buffer (RPMI 1640 containing 0.17 mg/ml liberase, 30 mg/ml DNase) for 30-35 minutes at 37°C temperature with gentle shaking. Solutions were then passed through 100-micron and 40-micron cell strainers to collect single-cell suspensions. Suspensions were centrifuged at 500g for 5 minutes to pellet cells, followed by resuspension into 300 ml FACS buffer (2% FBS in PBS). From the cell suspensions, ILCs were enriched using a mouse Lineage cell depletion kit (Milteny Biotec) following the manufacturer's protocol. Library was prepared using manufaturer's protocol (10x genomics, single cell 3' protocol)).In brief, cells were passed through Chromium Next GEM chip G with barcoded gel beads to generate GEM (Gel Beads-in-emulsion). After that, barcoded cDNA was generated in GEMs. GEMs were then broken, and cDNA was cleaned up and amplified for the library preparation. Sequencing was performed using the P3-100 cycle kit.